The present invention relates to a peptide factor isolated from steroid-treated monocytes. More particularly the invention relates to a peptide factor which can be used to replace steroid therapy.
Steroids are effectively used for anti-inflammatory diseases, such as asthma, eczema, allergic reactions, and rheumatic diseases such as rheumatoid arthritis. However, steroids have serious side effects and are therefore only used in cases where non-steroidal anti-inflammatory drugs are not effective.
Monocytes are important immune effector cells that play a fundamental role in cellular immunity. In addition to their antigen-presenting and phagocytic activities at the sites of inflammation, peripheral blood mononuclear cells are also involved in the synthesis and release of a variety of pro-inflammatory enzymes and polypeptide cytokines which modulate neutrophil responses. The production of these components can be suppressed by glucocorticoids and this has been suggested as the basis for their anti-inflammatory action.
The effect of steroid-induced factors on neutrophil migration is primarily of interest in elucidating anti-inflammatory mechanisms. Corticosteroids down regulate the synthesis of many pro-inflammatory mediators (Lew et al 1988; Almawi et al 1991; Standford et al 1992) but some of their actions can be interpreted in terms of up-regulation of anti-inflammatory mediators.
The neutrophil migration stimulating activity of steroid induced factors suggests that dispersive locomotion tends to prevent cells collecting at a focus and this may be important in terminating inflammatory responses.
Stevenson (1973, 1974, 1978) demonstrated that human monocytes when incubated in the presence of antiinflammatory corticosteroids released a protease sensitive factor that enhanced the migration of neutrophils from a cell pellet contained in a short capillary tube.
Later studies demonstrated that the phenomenon of stimulated neutrophil migration was also observed with leucocytes from patients receiving steroid therapy.
Recently, Chettibi et al (1993, 1994) have investigated the steroid induced stimulatory effect on neutrophil migration using an automated cell tracking assay enabling study of the behaviour of cells migrating on protein-coated glass coverslip.
These Studies Determined:
1. Steroid-treated monocyte supernatant (STMS) causes a dramatic increase in the speed of locomotion of human neutrophils and a significant decrease in their adhesion to protein-coated glass. In contrast, control monocyte supernatants have a smaller effect on the speed of locomotion, but cause a large increase in adhesiveness.
2. The supernatant activity was produced equally well in the presence or absence of serum after 24 h culture at 37xc2x0 C. with 10xe2x88x926M dexamethasone.
3. The effect of the steroid-treated monocyte supernatant on the speed of locomotion of human peripheral blood neutrophils was not altered by rabbit polyclonal antisera against lipocortins 1-6.
4. Rabbit anti-interleukin-8 antibody which blocked the effect of IL-8 on the speed of locomotion of neutrophils did not antagonize the locomotion stimulating action of steroid-treated monocyte supernatant.
5. The exocellular release of this factor(s) by human mononuclear leucocytes suggests that it may be an in vivo mediator of the anti-inflammatory effect of glucocorticoids.
However, there is no disclosure of what the active agent(s) in STMS might be.
Huff T. et al. (1995) and Heintz D. et al. (1994) describe studies involving beta-thymosins and how they interact with G-actin in a biomolecular complex and inhibit the polymerisation to F-actin under high salt conditions. The oxidised form of thymosin xcex24 is disclosed as inhibiting actin polymerisation, however, only at a 20-fold higher concentration than thymosin xcex24. Neither document however implicates any medical role for oxidised thymosin xcex24. In fact the papers appear to teach away from a positive role for oxidised thymosin xcex24.
U.S. Pat. No. 5,578,570 (Goldstein et al.) discloses a method of treating septic shock by administering thymosin xcex24. There is no disclosure however of oxidised thymosin xcex24 or suggestion that this may have a role in treating septic shock.
It is an object of the present invention to provide a replacement to steroid therapy.
The present invention is based in part on the observations by the present inventors that the factor associated with neutrophil locomotion is an oxidised form goof thymosin xcex24.
According to a first aspect the present invention provides use of oxidised thymosin xcex24 or physiologically active variant thereof in therapy.
Typically oxidised thymosin xcex24 is a form of thymosin xcex24 in which a methionine residue, 6 amino acids from the N-terminus, (Met6), is oxidised such that the residue is converted to methionine sulphoxide. Moreover, the methionine residue (Met6) may be further oxidised to the methionine sulphone and this as such is also encompassed by the present invention. Other modifications of the methionine residue may also be evisaged, such as complexing the sulphur with metals, which may result in an active form of thymosin xcex24 similar to the oxidised form described herein.
It is understood that the oxidised thymosin xcex24 may be obtained for example by reacting native thymosin xcex24 under oxidising conditions, for example by treating with hydrogen peroxide, to form oxidised thymosin xcex24. Thus native thymosin xcex24 may first be obtained and thereafter oxidised to the oxidised form.
It has been observed that samples of native thymosin xcex24 may contain low levels, such as 10%, of oxidised thymosin xcex24 thought to be as a result of auto-oxidation. The present inventors however are the first to associate the oxidised form of thymosin xcex24 with a physiological activity. Generally speaking therefore the present invention provides the use of purified oxidised thymosin xcex24. Typically the present invention provides use of preparations of purified oxidised thymosin xcex24 which comprise at least 30%, preferably 60%, more preferably 80%, most preferably 90%, oxidised thymosin xcex24 with the residual portion accounting for non-oxidised thymosin xcex24. Preferably however the preparations of oxidised thymosin xcex24 comprise substantially all oxidised thymosin xcex24 (ie. substantially no non-oxidised thymosin xcex24).
Thymosin xcex24 in an oxidised or non-oxidised form may be obtained from any suitable source, for example from steroid treated monocytes. Moreover, the thymosin xcex24 may be derived from any suitable species, but is typically of mammalian origin, such as bovine, equine, murine or human origin. It is to be noted that bovine, equine, murine, rat and human thymosin xcex24 are all identical in sequence. Thus, for example, bovine thymosin xcex24 may provide a suitable source of thymosin xcex24 for subsequent oxidation and administration to other species, such as humans.
It is understood that physiologically active variants of the oxidised thymosin xcex24 are variants which display the same or similar physiological properties as the oxidised thymosin xcex24. It is to be preferred that such variants would include the oxidised methionine, but may be truncated, deleted or mutated forms thereof.
It will be understood that for the particular oxidised thymosin xcex24 embraced herein, variations (natural or otherwise) can exist. These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. All such derivatives are included within the scope of this invention provided that the derivatives are physiologically active (ie. display oxidised thymosin xcex24 activity as defined herein). For example, for the purpose of the present invention conservative replacements may be made between amino acids, within the following groups:
(I) alanine, serine and threonine;
(II) glutamic acid and aspartic acid;
(III) arginine and lysine;
(IV) asparagine and glutamine;
(V) isoleucine, leucine and valine;
(VI) phenylalanine, tyrosine and tryptophan.
(VII) methionine and other methionine analogues
(VIII) methionine and other methionine analogues where the sulphur is replaced by Group VIB elements (e.g. Selenium, Tellurium, Polonium).
(IX) oxidised methionine and other oxidised methionine analogues (e.g. Group VIB analogues, methionine sulphoximine).
(X) methionine and other sulphur-containing amino acids (e.g. cysteine) including their oxidised analgoues.
Early molecular modelling studies suggest that the methionine residue (met-6) is at the top of one of three helices in the peptide. Molecular modelling should help identify a shorter peptide which may have the activity observed for STMS and oxidised thymosin xcex24 and would be a preferred molecule to use in preparing pharmaceuticals with anti-inflammatory activity.
Indeed this may assist in the development of peptide mimetics which display the same physiological function as the oxidised thymosin xcex24.
Moreover, it may be possible to increase the half life of oxidised thymosin xcex24 or physiologically active variants thereof by use of appropriate chemical modification (eg. acetylation) or use of D amino acids.
The isolated oxidised thymosin xcex24 may have a blocked N-terminal.
According to the present invention there is also provided a synthetic oxidised thymosin xcex24 comprising the peptide sequence of thymosin xcex24 in oxidised form or physiologically active variant thereof.
The synthetic oxidised thymosin xcex24 may be modified and/or amino acid substituted as described above, as long as the physiological activity remains. For example seleno-methionine could be introduced in place of methionine and oxidised in the same manner.
The invention further provides the use of an oxidised peptide as described herein in the preparation of a medicament for the treatment of a chronic or acute inflammatory condition. Such inflammatory conditions include Inflammatory Arthropathies such as Rheumatoid arthritis, Psoriatic arthritis, Crystal arthritis, Reactive arthritis, Ankylosing spondylitis, Infectious arthritis, Juvenile chronic arthritis; Connective Tissue Diseases, such as Systemic Lupus Erythematosis, Sjogren""s Syndrome, Polymyalgia Rheumatica, Cranial arteritis; Vasculitic Syndromes, such as Wegener""s Granulomatosis, Polyarteritis Nodosa, Churg Strauss Syndrome; Respiratory Diseases, such as Asthma, Chronic Obstructive Pulmonary Disease, Fibrosing Alveolitis, Hypersensitivity Pneumonitis, Sarcoidosis, Allergic aspergillosis, Cryptogenic pulmonary eosinophilia, Bronchiolitis obliterans organising pneumonia; Dermatological Diseases, such as Inflammatory dermatosis including psoriasis, Eczema, Urticaria; Gastro-intestinal Diseases, such as Ulcerative Colitis, Crohn""s Disease, Lupoid hepatitis; Haematological Disease, such as Haemolytic anaemia, Idiopathic Thrombocytopenic Purpura, Multiple Myeloma, Lymphoma/leukaemia; Transplantation/Prosthetics, such as Graft rejection, Graft versus host disease, Tissue reaction to implanted prostheses; and Infections, such as Tuberculosis, Malaria Pneumocystis carinii pneumonia, Leprosy.
Moreover, oxidised thymosin xcex24 may be administered in conjunction with other drugs, eg. cytokines such as interferon which may induce an inflammatory response as a side effect. Thus, in one aspect oxidised thymosin xcex24 may serve to minimise or reduce physiological or disease states which are characterised in part by inappropriate inflammation.
Additionally, it should be appreciated that the uses of oxidised thymosin xcex24 mentioned above do not only extend to human conditions. Thus, oxidised thymosin xcex24 may be used in the treatment of animals such as cats, dogs, horses, cows, sheep, pigs and goats with similar conditions to those mentioned above.
The present invention further provides the use of oxidised thymosin xcex24 in the preparation of a medicament for the treatment of septic shock. Typically the oxidised thymosin xcex24 is in a purified form as described above.
The invention further provides a pharmaceutical composition comprising oxidised thymosin xcex24 as described herein.
The invention further provides use of a nucleotide molecule having a sequence capable of encoding thymosin xcex24 as described herein for subsequently preparing oxidised thymosin xcex24.
In a particular embodiment the invention provides the use of a vector or vectors comprising the nucleotide molecule in the preparation of oxidised thymosin xcex24 and trancated, deleted and mutated forms thereof as described herein.
Alternatively the present invention provides the use of a vector or vectors comprising the nucleotide molecule in the preparation of a medicament comprising oxidised thymosin xcex24 and trancated, deleted and mutated forms thereof for the treatment of a inflammatory condition.
The use of oxidised thymosin xcex24 as described herein in place of steroid treatment will alleviate the side effects which are normally associated with the use of steroids.
The oxidised thymosin xcex24 can be used for treatment of patients where non steroidal anti inflammatory drugs are currently used as an alternative to steroids because of the risks of side-effects.
Use of highly purified oxidised thymosin xcex24 or of synthetic or expressed thymosin xcex24 which is subsequently oxidised will be safe and reliable, since it will generally not be foreign to the body to which it is being administered.
Accurate amounts can be administered.
The amount of oxidised thymosin xcex24 required to be effective in a treatment will, of course, vary and is ultimately at the discretion of the medical or veterinary practitioner. The factors to be considered include the condition being treated, the route of administration, and nature of the formulation, the recipients body weight, surface area, age and general condition, and the particular compound to be administered. A suitable effective dose may lie in the range of about 0.001 to about 120 mg/kg bodyweight, e.g. 0.01 to about 120 mg/kg body weight, preferably in the range of about 0.01 to 50 mg/kg, for example 0.05 to 20 mg/kg. The total daily dose may be given as a single dose, multiple doses, e.g., two to six times per day or by intravenous infusion for selected duration. For example, for a 75 kg mammal (e.g. a human) the dose range may be about 8 to 9000 mg per day, and a typical dose could be about 50 mg per day. If discrete multiple doses are indicated treatment might typically be 15 mg of oxidised thymosin xcex24 given up to 4 times per day.
Whilst it is possible for the active compound to be administered alone, it is preferable to present the active compound in a pharmaceutical formulation. Formulations of the present invention, for medical use, comprise oxidised thymosin xcex24, or a salt thereof together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The carrier(s) should be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.
The present invention, therefore, further provides a pharmaceutical formulation comprising oxidised thymosin xcex24 or a pharmaceutically acceptable salt or physiologically functional derivative thereof together with a pharmaceutically acceptable carrier therefor.
There is also provided a method for the preparation of a pharmaceutical formulation comprising bringing into association oxidised thymosin xcex24 or a pharmaceutically acceptable salt or physiologically functional derivative thereof, and a pharmaceutically acceptable carrier therefor.
Formulations according to the present invention include those suitable for oral, nasal, topical, vaginal, rectal or parenteral (including subcutaneous, intraarthrodial (ie. within joints) intramuscular and intravenous) administration including biolistic eg. Powderject(copyright) administration. Preferred formulations are those suitable for oral, topical or parenteral administration.
The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active compound into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier or a finely divided solid carrier or both and then, if necessary, shaping the product into desired formulations.
Formulations of the present invention suitable for oral administration may be presented as discrete units as capsules, cachets, tablets, lozenges, comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier. Each formulation generally contains a predetermined amount of the active compound; as a powder or granules; or a solution or suspension in an aqueous or non-aqueous liquid such as a syrup, an elixir, an emulsion or draught and the like.
A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed, tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form such as a powder or granules, optionally mixed with a binder, (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile.
A syrup may be made adding the active compound to a concentrated, aqueous solution of a sugar, for example sucrose, to which may also be added any accessory ingredients. Such accessory ingredient(s) may include flavourings, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredients, such as a polyhydric alcohol for example glycerol or sorbitol.
Formulations for rectal administration may be presented as a suppository with a conventional carrier such as cocoa butter.
Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably isotonic with the blood of the recipient. Such formulations suitably comprise a solution of a pharmaceutically and pharmacologically acceptable salt of oxidised thymosin xcex24, that is isotonic with the blood of the recipient.
Useful formulations also comprise concentrated solutions or solids containing oxidised thymosin xcex24, which upon dilution with an appropriate solvent give a solution for parental administration as above.
The oxidised thymosin xcex24 or physiologically active variant thereof disclosed herein may be administered to the lungs of a subject by any suitable means, but are preferably administered by generating an aerosol comprised of respirable particles, the respirable particles comprised of the active compound, which particles the subject inhales (i.e., by inhalation administration). The respirable particles may be liquid or solid.
Particles comprised of oxidised thymosin xcex24 for practising the present invention should include particles of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. In general, particles ranging from about 0.5 to 10 microns in size (more particularly, less than about 5 microns in size) are respirable. Particles of non-respirable size which are included in the aerosol tend to deposit in the throat and be swallowed, and the quantity of non-respirable particles in the aerosol is preferably minimized. For nasal administration, a particle size in the range of 10-500 xcexcm is preferred to ensure retention in the nasal cavity.
Liquid pharmaceutical compositions or oxidised thymosin xcex24 for producing an aerosol can be prepared by combining the oxidised thymosin xcex24 with a suitable vehicle, such as sterile pyrogen free water. Solid particulate compositions containing respirable dry particles of micronized oxidised thymosin xcex24 may be prepared by grinding dry oxidised thymosin xcex24 with a mortar and pestle, and then passing the micronized composition through a 400 mesh screen to break up or separate out large agglomerates. A solid particulate composition comprised of the oxidised thymosin xcex24 may optionally contain a dispersant which serves to facilitate the formation of an aerosol. A suitable dispersant is lactose, which may be blended with the oxidised thymosin xcex24 in any suitable ratio (e.g., a 1 to 1 ratio by weight).
Aerosols of liquid particles comprising the oxidised thymosin xcex24 may be produced by any suitable means, such as with a nebulizer. See, e.g., U.S. Pat. No. 4,501,729. Nebulizers are commercially available devices which transform solutions or suspensions of the oxidised thymosin xcex24 into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. Suitable compositions for use in nebulizers consist of the oxidised thymosin xcex24 in a liquid carrier, the oxidised thymosin xcex24 comprising up to 40% w/w of the compositions, but preferably less than 20% w/w. the carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride. Optional additives include preservatives if the composition is not prepared sterile, for example, methyl hydroxybenzoate, antioxidants, flavouring agents, volatile oils, buffering agents and surfactants.
Aerosols of solid particles comprising the oxidised thymosin xcex24 may likewise be produced with an solid particulate medicament aerosol generator. Aerosol generators for administering solid particulate medicaments to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a medicament at a rate suitable for human administration. Examples of such aerosol generators include metered dose inhalers and insufflators.
For inflammation of external tissues, e.g. skin, the formulations are preferably applied as a topical ointment or cream containing the active ingredient in an amount of, for example, 0.075 to 20% w/w, preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. When formulated in an ointment, the active ingredients may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base.
If desired, the aqueous phase of the cream may include, for example, at least 30% w/w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glyercol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulphoxide and related analogues.
The oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyle alcohol, glycerol mono-stearate and sodium lauryl sulphate.
The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono-or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
In addition to the aforementioned ingredients, the formulations of this invention may further include one or more accessory ingredient(s) selected from diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like.
The following examples describe the purification of the peptide factor and the characterisation of partially purified factor and steroid-treated monocyte supernatant. Later examples describe the characterisation of purified factor as oxidised thymosin and activity of the oxidised thymosin xcex24.